Mechanisms and applications of mesenchymal stem cell exosomes in enhancing fertility

Abstract

Infertility is an increasingly prevalent global health issue affecting millions of couples. It can arise from various factors, including hormonal imbalances, reproductive disorders, and anatomical abnormalities. While numerous treatment options exist, such as assisted reproductive technologies, these approaches often entail significant costs and have limited success rates. In this paper, we review the role of mesenchymal stem cell exosomes (MSC-Exos) in the treatment of infertility, with a particular focus on their potential to enhance fertility. We herein aimed to integrate the latest research progress in this field and emphasize the importance of MSC-Exos as a potential treatment for infertility.

Introduction

Globally, infertility affects approximately 15% of couples seeking to conceive. A total of 33–41% of infertility cases are attributed solely to female factors, 25–39% to male factors, and 9–39% to a combination of both male and female factors. Infertility can lead to significant psychological distress for individuals, as well as substantial suffering and economic burdens for the community [1].Infertility is defined as the inability of a couple to conceive after one year of regular, unprotected intercourse. This condition can arise from various factors that include both female and male factors, hormonal imbalances, reproductive disorders, anatomical abnormalities, and lifestyle influences [2]. Traditional assisted reproductive technologies (ARTs), such as intrauterine insemination (IUI), in vitro fertilization-embryo transfer (IVF-ET), and intracytoplasmic sperm injection (ICSI) have long served as the primary solutions for addressing infertility [3, 4].

An increasing number of infertile couples are opting for ART as a treatment option [5]; however, there are several risks associated with this, including ovarian hyperstimulation syndrome (OHSS), bleeding, and infections following ART procedures. While these complications are infrequent, they can pose life-threatening risks [6].In addition to ART, alternative therapies such as hormonal therapy, surgery, and lifestyle modifications exist [7], but these approaches are often invasive and costly [8].

To address the limitations of these therapies, there is an urgent need to explore new treatment options, and stem cell therapy represents a promising avenue for treating reproductive disorders. Mesenchymal stem cells (MSCs) exhibit a remarkable ability to differentiate into various cell types with differing immunomodulatory properties and anti-inflammatory effects. They can inhibit fibrogenesis, possess antioxidant and regenerative properties, and are used in tissue repair and the treatment of various diseases. Although MSCs offer novel therapeutic avenues for patients with tubal infertility and diminished ovarian reserve by repairing endometrial damage and regulating the ovarian immune microenvironment [9], their clinical application still faces several challenges. These include the susceptibility of biological activity to the in vitro environment, difficulties in cell quantification, functional decline during transportation, and the need for immunological matching between donors and recipients—all of which increase the complexity of treatment. To overcome these limitations, MSC-derived exosomes (MSC-Exos) have emerged as a novel cell-free therapeutic strategy [10]. As carriers of intercellular signal transduction, MSC-Exos not only possess the reparative and immunomodulatory functions of MSCs in treating infertility, but they also offer the advantages of stable biological activity, and ease of standardized extraction, while also lacking, the requirement for immunological matching. Hence, their potential role in enhancing fertility has become a focus in research in the field of reproductive medicine in recent years.

Infertility

Infertility affects many couples of reproductive age worldwide. According to the World Health Organization, approximately 15% of couples of reproductive age are affected by infertility, with its prevalence increasing annually [1]. Female infertility encompasses numerous conditions, including ovulatory disorders [11], uterine or cervical abnormalities [12], tubal injury or obstruction [13], endometriosis, and cancer and its treatment. These diseases are not only directly related to infertility, but they are also often accompanied by symptoms such as dysmenorrhea, menstrual disorders, and chronic pelvic pain, all of which can seriously affect a patient’s quality of life. The etiology of male infertility—although significantly distinct from that of females—is equally critical in its impact on reproductive outcomes. These conditions include the inappropriate production or malfunction of sperm, sperm-transport issues [14], overexposure to specific environmental factors, and cancer-related effects [15]. In this review, we scrutinize the pathomechanisms underlying common infertility disorders in both men and women and the patterns of their interference with fertility, and also lay the groundwork for the subsequent exploration of therapies such as MSC-Exos (Fig. 1).

Fig. 1.

Causes of infertility. Abbreviations: PCOS, polycystic ovary syndrome; HPO axis, hypothalamic–pituitary–ovarian axis; POI, premature ovarian insufficiency; CS, chronic salpingitis; TE, thin endometrium; EM, endometriosis; IUA, intrauterine adhesions. Created in BioRender by Wang, Y, (2025) (https://BioRender.com/w0v5cw8)

Functional disorders of the ovaries

Ovulatory disorders are the first factor in infertility in women of reproductive age, with approximately 40% of women being suffering from them [11]. Among these disorders, polycystic ovary syndrome (PCOS) and premature ovarian insufficiency (POI) comprise the major diseases of ovarian function.

Ovulatory disorders are most common in PCOS, and the prevalence of infertility in patients with PCOS has reached 70%, seriously affecting overall fertility [16]. PCOS is characterized by chronic anovulation and hyperandrogenemia, with clinical manifestations that include menstrual disorders, hirsutism, acne, and polycystic changes in the ovaries. Furthermore, PCOS is often associated with metabolic disorders such as obesity, insulin resistance, and dyslipidemia. Its pathogenesis remains unknown, and local cytokines in the ovary, immunological and genetic factors, and perturbations of the hypothalamic-pituitary-ovarian axis may be responsible for its development [17]. Premature ovarian insufficiency (POI), also known as premature ovarian failure (POF), is an ovarian dysfunction that occurs in women before the age of 40 years and is manifested by amenorrhea or oligomenorrhoea, elevated gonadotropins, and reduced estradiol (E2) concentrations. POI greatly reduces a woman’s quality of life; increases her risk of osteoporosis, cardiovascular disease, and neurological disorders; and necessitates monitoring and treatment [18]. The incidence of POI has risen in recent years, attaining a global prevalence of 3.5%. Its common causes include genetic, immunological, infectious, medical, and environmental factors. The majority of POI cases are idiopathic with no identifiable risk factors, while the remaining cases are defined as medically induced POI. Exposure to chemotherapeutic drugs is the most common cause of medically induced POI and accounts for approximately half (20–80%) of new cases in women of childbearing age. POIs are frequently reported in cured cancer survivors, and the incidence of POIs has increased with the recent developments in chemotherapeutic agents [19].

Dysfunctional uterine diseases

Appropriate embryonic implantation is a highly controlled process that requires a receptive endometrium [20]. Uterine pathologies can therefore lead to a reduced rate of embryonic implantation in women of childbearing age, including endometrial polyps, uterine fibroids, thin endometrium (TE), and intrauterine adhesions (IUA) [12]. Although there is no standardized protocol for the diagnosis of TE, the recommended minimal critical endometrial thickness in embryo transfer is 7 mm [21], with a thickness above 9 mm predicting higher rates of implantation and clinical pregnancy. For every 1 mm decrease in endometrial thickness, there is an increase in associated pregnancy complications such as early spontaneous abortion, preterm delivery, low birthweight, hypertensive disorders of pregnancy, placental abruption, and ectopic pregnancy in women who conceived with IVF-ET [22]. IUA is defined as an injury to the basal layer of the endometrium, with the formation of bands of fibrous tissue on the wall of the uterine cavity that results in partial or complete occlusion of the uterine cavity. IUA is also known as Asherman syndrome (AS) and manifests in the form of menstrual abnormalities, cyclic lower abdominal pain, recurrent miscarriages, and secondary infertility [23]. Intrauterine surgery or infection is also a high-risk factor for adhesion formation [24]. The prevalence of IUA varies according to the type of injury, ranging from 16% to 24% in women undergoing abortion curettage and from 31% to 45% after hysteroscopic myomectomy. The process of endometrial injury repair involves an inflammatory phase, a tissue-forming phase, and a tissue-reconstruction phase. Although the mechanisms underlying injury repair are unknown, it may involve hypoxia, reduced neovascularization, and changes in the expression of adhesion-related cytokines [25].

Tubal infertility

Several factors such as infection, surgery, ectopic pregnancy, and endometriosis can precipitate chronic salpingitis (CS) or pelvic inflammatory disease (PID), with tubal mucosal damage and structural collapse that result in scarring of the tubes and mild adhesions, complete blockage, or the absence of fallopian tubes. These effects ultimately lead to tubal infertility [13].

Endometriosis (EM) is a chronic inflammatory disease in which endometrial tissue (glands and mesenchyme) appears outside the body of the uterus, with an overall prevalence of 0.8–6%. However, a higher prevalence of approximately 20–50% has been observed in women with low fertility [26]. This may be due to anatomical deformities caused by conditions that range from adhesions and fibrosis to endocrine abnormalities and immune dysfunction; the mechanisms underlying the various pathophysiological disturbances are as yet unknown [27]. Approximately 50% of women with mild EM will conceive without treatment, whereas only 25% of women with moderate EM will conceive spontaneously, and few women with severe EM will conceive spontaneously [28]. Neisseria gonorrhoeae and Chlamydia trachomatis, both of which are Gram-negative organisms and exclusive human pathogens, are the most common causes of sexually transmitted diseases (STDs) [29], and infection with them can potentially result in chronic PID.

Sperm-related disorders

Infertility may be induced by a variety of mechanisms such as gametocyte damage, decreased sperm quality and obstruction of the male reproductive tract [14]. Common causes include varicocele, cryptorchidism, infections, obstructive lesions, cystic fibrosis, trauma, and tumors; oxidative stress has also been identified as a causative factor in a recent study [30].

Male reproductive tract infections

A prominent element of male infertility is reproductive tract infections. The most common sexually transmitted microorganism is C. trachomatis, but its role in male infertility is controversial [31]. Nevertheless, there is evidence that C. trachomatis infection creates an inflammatory response that can lead to obstruction of the seminal vasculature [32]. Additionally, semen from C. trachomatis-infected patients typically has reduced semen volume, decreased sperm motility, alterations in sperm concentration, and a modified pH [15]. Many microbial sexually transmitted infections (STIs), such as that associated with C. trachomatis are asymptomatic in low-fertility men [32] and should be screened for and promptly treated to prevent damage to male fertility [33]. This STI can also lead to elevated sperm matrix metalloproteinase (MMP) and caspase-3 activation, ultimately inducing apoptosis [34].

Different sources of MSCs and their role in infertility

MSCs have received increasing attention for their potential therapeutic utility in various female reproductive disorders. They are capable of differentiating into various cell types including adipocytes, osteoblasts and chondrocytes under cell culture conditions [35]. MSCs are classified according to their origin, and their role in infertility treatment is summarized in what follows (Fig. 2).

Fig. 2.

Infertility conditions treatable with different types of MSCs. Abbreviations: MSCs, mesenchymal stem cells; BM-MSCs, bone marrow-derived mesenchymal stem cells; ADSCs, adipose-derived mesenchymal stem cells; UC-MSCs, umbilical cord-derived mesenchymal stem cells; AD-MSCs, amnion-derived mesenchymal stem cells; PSC-MSCs, pluripotent stem cell-derived mesenchymal stem-like cells; PMSCs, placenta-derived mesenchymal stem cells; AFSCs, amniotic fluid-derived mesenchymal stem cells; MenSCs, menstrual blood-derived mesenchymal stem cells; PCOS, polycystic ovary syndrome; POI, premature ovarian insufficiency; CS, chronic salpingitis; TE, thin endometrium; EM, endometriosis; IUA, intrauterine adhesions. Created in BioRender by Wang, Y, (2025) ( https://BioRender.com/9achrg5)

Bone marrow-derived mesenchymal stem cells (BM-MSCs)

BM-MSCs are a heterogeneous population of cells that support hematopoiesis. They were first isolated from nucleated bone marrow cells by Owen and Friedenstein in 1988 [36]. BM-MSCs possess the ability to differentiate into chondrocytes, osteoblasts, and adipocytes, and they account for a small proportion of the nucleated bone marrow cell population [37]. These MSCs can also differentiate into endometrium [38], endothelial cells [39], and granulosa cells (GCs) [40] and can be adopted to treat infertility to a degree.

With regard to improving ovarian function, although chemotherapy is an effective means of cancer treatment, it often causes ovarian dysfunction and infertility. Several studies have shown that BM-MSCs significantly improve chemotherapy-induced ovarian dysfunction through the secretion of angiogenic factors and growth factors. Lu et al. found [40] that the combination of BM-MSCs and moxibustion (BM-MSC-MOX) reduced the levels of reactive oxygen species (ROS) in ovarian tissues and simultaneously upregulated the MMP and adenosine triphosphate (ATP) levels in ovarian GCs, improving mitochondrial function. In addition, it further facilitated mitochondrial function by downregulating dynamin-related protein 1 (Drp1) and PTEN-induced putative kinase 1 (PTEN-induced), among other mitochondrial molecules. In addition, by downregulating the expression of mitochondrial autophagy-related proteins such as Drp1, PTEN-induced putative kinase 1 (PINK1), and parkin RBR E3 ubiquitin-protein ligase (Parkin), the combination inhibited excessive mitochondrial autophagy, attenuated mitochondrial damage, and effectively repaired cyclophosphamide-induced ovarian damage. Badawy et al. [41] ascertained that mice treated with BM-MSCs were more susceptible to autophagy than mice treated with BM-MSCs. Additionally, E2 levels declined and follicle-stimulating hormone (FSH) levels increased in their MSC-treated mouse model, but both hormones recovered to near-normal levels, accompanied by the formation of new primordial follicles and the effective treatment of POI.

In endometrial repair, BM-MSCs migrate to the uterus and promote regeneration of injured endometrium. Santamaria et al. [42] demonstrated that the use of autologous peripheral blood CD133+-labelled BM-MSCs increased menstrual flow, menstrual period length, endometrial thickness, and angiogenic processes while decreasing the uterine adhesion score, thereby effectively treating IUA. Cervelló et al. [43] determined that BM-MSCs labelled with CD133 + were also able to induce pericyte proliferation and increase endometrial thickness by targeting angiogenesis and secreting growth factors such as thrombospondin-1 (TSP-1) and insulin-like growth factor-1 (IGF-1). Additional studies have revealed that BM-MSC replacement therapy improves endothelial dysfunction and treats infertility in patients with TE and IUA by enhancing endothelial tolerance, promoting angiogenesis, and inhibiting fibrosis [44, 45]. In male infertility, Khanmohammadi et al. [46] found that BM-MSCs enriched in Sertoli cell-conditioned medium (SCCM) effectively differentiated into male germ-like cells and mitigated non-obstructive azoospermia. Zhang et al. [47] discovered that in vitro-induced BM-MSCs expressed spermatogonial genes and protein markers and differentiated into spermatogonia-like cells; this mechanism could then promote the restoration of endogenous fertility, providing a potential treatment for patients with non-obstructive infertility as a consequence of cancer chemotherapy. Liu et al. [48] modified BM-MSCs by overexpressing miRNA-145 to increase the content of smooth muscle in the penile tissues of aged rats, effectively attenuating the erectile dysfunction characteristic of aged male rats.

Adipose-derived mesenchymal stem cells (ADSCs)

ADSCs are an emerging source of MSCs with a wide range of applications in tissue regeneration [49]. ADSCs possess unique advantages over other sources; for example, they can be easily obtained by minimally invasive methods and exhibit immunosuppressive properties. Their ability to differentiate into adipocytes, osteoblasts, chondrocytes, neuronal cells, cardiomyocytes, myocytes, vascular endothelial cells, and hepatocytes (congruent with other MSCs) reflects a wide range of potential for clinical applications [50, 51].

ADSC-based stem cell therapy can upregulate vascular endothelial growth factor (VEGF) expression in rats, promote neovascularization and improve ovarian transplantation [52]. It also repairs chemotherapy-damaged ovarian function in mice, increasing the number of blood vessels and follicles [53]. Su et al. [54] ascertained that compared with ADSC treatment alone, ADSCs combined with collagen scaffolds elevated the numbers of ovarian cells and follicles. Additional work by these authors revealed that the combination of ADSCs and collagen scaffolds improved short-term retention in the ovaries of POI rats and facilitated the restoration of ovarian function in the long term. ADSCs increased follicle number, promoted ovulation, and reduced GC death in a cyclophosphamide (CTX)-induced POI model [55]. In addition, ADSCs in combination with estrogen upregulated the expression of VEGF and IGF-1, downregulated miR-98 and miR-199a, reduced endometrial fibrosis, and enhanced endometrial regeneration in an IUA rat model [56]. However, Abomaray et al. [57] demonstrated that allogeneic ADSCs isolated from EM patients induced ectopic endometrial tissue growth and therefore did not recommend these cells for use in the treatment of EM.

Lin et al. [58] suggested that ADSCs played a key role in erectile dysfunction and male infertility. However, experimental evidence is currently lacking.

Menstrual blood-derived mesenchymal stem cells (MenSCs)

MenSCs are an emerging source of MSCs and have attracted considerable attention since their discovery in 2007. They are collected from menstrual blood by non-invasive techniques and possess unique stem cell properties, thus providing a rich source of replacement cells for tissue regeneration [59]. MenSCs are not only easily obtained and manifest a high proliferative capacity, but autoimmune rejection has not yet been observed [60].

In terms of repairing ovarian function, MenSCs can reduce the death of ovarian mesenchymal GCs by preserving them, thereby improving the damaged immune system through paracrine mechanisms so as to restore the reserves of primordial and growing follicles, and recapitulating ovarian function in POI mice [61]. In another study, MenSCs were shown to upregulate the level of heparin-binding growth factor 2 (FGF2) and to promote the restoration of ovarian structure and function [62]. Several studies have revealed that MenSCs promote the regeneration of damaged endometrium and inhibit inflammation and fibrosis by regulating the Wnt family member 5a (Wnt5a), growth differentiation factor 5 (Gdf5), and Hippo signaling pathways [63]. For example, Domnina et al. [64] determined that MenSCs could enhance fertility by promoting angiogenesis and secreting anti-inflammatory factors in a rat model of IUA. In 2018, for the first time, Zheng et al. [65] demonstrated that MenSCs differentiated into endometrial cells in vitro and rebuilt the endometrial tissues in response to estrogen and progesterone in an animal model. Their combined hormonal treatment also improved the endometrial structure and fertility of women with IUA [66]. Subpopulations of cells with characteristics of MenSCs are present in the endometrium, but MenSCs originating from ectopic endometrial foci may be involved in the pathogenesis of EM [67].

Lu et al. [68] declared that the expression of 3β-hydroxysteroid dehydrogenase (3β-HSD, which primarily catalyzes the conversion of androstenedione to testosterone and is the rate-limiting enzyme for steroid biosynthesis) was significantly upregulated in testicular tissues of MenSC-treated type 1 diabetic (T1D) mice. This suggests that MSC transplantation can improve reproductive function in male T1D mice by promoting testosterone biosynthesis. MenSCs also significantly upregulated the expression of pro-angiogenic factors, reduced the infiltration of lymphocytes into penile tissues, and improved penile function in mice with T1D.

Umbilical cord-derived mesenchymal stem cells (UC-MSCs)

UC-MSCs are a rich source of MSCs that express stem cell-specific markers and can differentiate into a wide range of mesodermal cells for tissue repair and immunomodulation. These MSCs possess significant advantages such as rapid self-renewal, low oncogenicity, low immunogenicity, and non-invasive acquisition, and they have become a preferred source for stem cell transplantation [69]. UC-MSCs are routinely isolated from Wharton’s jelly, umbilical cord blood, or the perivascular tissue of the umbilical cord through established protocols, including enzymatic digestion (typically employing collagenase type II) and tissue explant methodologies [70].

In terms of repairing of ovarian function, Chen et al. [71] found that UC-MSCs attenuated cisplatin-induced POI by inhibiting iron death (ferroptosis), improving ovarian hormone levels, and decreasing the expression of the fibrosis-associated factors α-smooth muscle actin (α-SMA) and collagen type I (COL-I). Mohamed et al. [72] discovered that UC-MSCs upregulated anti-Müllerian hormone (AMH) and FSH receptor (FSHR) expression in GCs to inhibit apoptosis and follicular atresia in GCs and improved the functionality of ovaries injured by chemotherapy.

UC-MSCs have been demonstrated to activate primordial follicles, improve ovarian function, and reduce cell death in both animal and human models of POI [73, 74]. In addition, Li et al. [75] discovered that UC-MSCs secreted pro-angiogenic factors such as VEGF, hepatocyte growth factor (HGF), placental growth factor (PGF), and transforming growth factor-beta 1 (TGF-β1). UC-MSC transplantation restored dehydroepiandrosterone (DHEA)-induced ovarian function in PCOS model mice by downregulating interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and other inflammatory factors, reducing connective tissue growth factor (CTGF) levels; and downregulating the expression of other fibrosis-related genes [76].

In the field of endometrial regeneration, UC-MSCs can be used to repair endometrial tissues damaged by caesarean section scarring and can even differentiate into endometrial cells [77, 78]. Work by Zhang et al. [79] revealed that UC-MSCs improved endometrial injury and infertility by inhibiting inflammation, reducing fibrosis, promoting cellular proliferation, and enhancing the expression of vascular markers. Cao et al. [80] validated the positive effect of collagen stenting combined with UC-MSCs on endometrial tissue and infertility through a phase-I clinical trial. These authors also demonstrated that collagen scaffolds combined with UC-MSCs promoted endometrial regeneration in rats by inducing matrix metalloproteinase 9 (MMP9) expression. This treatment enhanced cellular proliferation and differentiation via the upregulation of von Willebrand factor (VWF), antigen KI-67 (Ki67), vimentin, and estrogen receptor alpha (ERα) gene expression—denoting therapeutic potential for recurrent IUA.

Liao et al. [81] found that UC-MSC reduced hydrops, macrophage infiltration, and IL-10 expression in the fallopian tubes induced anti-apoptotic effects by decreasing the expression level of cysteine-dependent aspartate-specific protease 3 (caspase-3). The application of UC-MSCs in a mouse model of chronic salpingitis resulted in a significant increase in pregnancy rate, and these effects were attributed to their anti-inflammatory and anti-apoptotic properties. Li et al. [82] showed that the concentration of TNF-α was significantly reduced in the group treated with UC-MSCs and that transplantation of UC-MSCs into New Zealand white rabbits with chronic salpingitis contributed to a partial restoration of fertility. UC-MSCs repaired the epithelial structure of the oviducts affected by chronic inflammation, reduced the level of inflammatory factors, and partially restored the secreted levels of oviductal glycoproteins.

Huang et al. [83] substantiated that UC-MSCs improved sperm quality and attenuated testicular damage in hypospermic rats by inhibiting excessive autophagy while activating the AKT/mTOR pathway. Additional studies showed that inhibition of the AKT/mTOR pathway partially reversed the therapeutic actions of UC-MSCs on olfactory receptor neuron-induced IUA in rats [84].

Role of other sources of MSCs in infertility

Amniotic fluid supports fetal growth by providing nutrients during embryonic development and pregnancy. Amniotic fluid-derived mesenchymal stem cells (AFSCs) comprise a novel source of stem cells free from the ethical controversy of embryonic stem cells, possess immune-modulating properties, are easily accessible, and regulate ovarian function primarily through paracrine signals (e.g., TGF-alpha [TGF-α], TGF-β, and VEGF) [85]. Although AFSCs do not directly differentiate into GCs in vivo, they can inhibit follicular atresia, maintain a good number of healthy follicles, and promote ovarian function in POI mouse models [86, 87]. Ibrahim et al. [88] ascertained that AFSCs played a potential therapeutic role in ameliorating leucovorin-induced azoospermia in adult rats by colonizing the basement membranes of damaged spermatogenic tubules, attenuating leucovorin-induced degeneration and oxidative damage, and inducing the re-expression of proliferating cell nuclear antigen (PCNA) in spermatocytes—leading to the restoration of spermatogenesis and the reemergence of spermatozoa.

Adipose-derived mesenchymal stem cells (AD-MSCs) constitute a potential cell source for regenerative medicine and are readily obtained from human adipose tissue through a non-invasive method. Per Li et al. [89], the therapeutic activity of AD-MSCs could be related to collagen secretion, and their action can be attributed to the fact that they exhibited higher telomerase activity and elevated pluripotent marker expression, as indicated by the augmented expression of transcription factor-like octamer-binding transcription factor 4 (OCT4) and NANOG homeobox (NANOG), improving cytokine production via the JAK/STAT pathway. AD-MSCs have been shown to exert a protective action on ovarian function in a chemotherapy-induced POI rat model. Ling et al. [90] determined that AD-MSCs improved ovarian function in chemotherapy-induced POI rats through a paracrine mechanism that the authors attributed to the secretion of growth factors such as FGF2, IGF-1, HGF, and VEGF by AD-MSCs—thus shielding the ovary by inhibiting apoptosis, promoting angiogenesis, and regulating the intrafollicular microenvironment surrounding the GCs.

In contrast to the paracrine mechanism of AD-MSCs, placenta-derived mesenchymal stem cells (PMSCs) mitigate GC apoptosis by suppressing the inositol-requiring enzyme 1 alpha(IRE1α) signaling pathway, thereby facilitating ovarian function recovery in autoimmune-induced POI mice [91]. Furthermore, PMSCs enhance ovarian function in ovariectomized rats by stimulating estrogen secretion and upregulating folliculogenesis-related gene expression [92]. Yin et al. [93] found that after transplantation of PMSCs, the treatment-induced diminution in serum TGF-β and increase in IFN-γ induced by recombinant zona pellucida glycoprotein 3 (pZP3) were reversed, and this was shown to be the result of a reversal of the treatment-induced increase in IFN-γ within PMSCs. This suggests that the recovery of ovarian function in POI mice is mediated by the regulation of regulatory T cells (Treg) and related cytokines produced by PMSCs after transplantation.

Pluripotent stem cell-derived mesenchymal stem-like cells (PSC-MSCs) are a novel source of tissue regeneration that are differentiated from pluripotent stem cells (PSCs) [94]. In 2010, Lian et al. demonstrated that PSC-MSCs could differentiate into osteoblasts. Additionally, MSCs are now well-known to differentiate into osteoblasts, adipocytes, and chondrocytes and to have pro-angiogenic properties [95]. PSC-MSCs demonstrate superior engraftment and immunomodulation over BM-MSCs, making them a viable therapeutic strategy for inflammation-associated infertility [96].

Different sources of MSC exosomes and their roles in infertility

MSCs primarily exert their therapeutic effects in infertility treatment through the paracrine secretion of cytokines and growth factors [97, 98], thereby promoting endometrial repair, ovarian function improvement, and immune modulation [41, 43, 52, 53]. However, their clinical translation faces several challenges, including low cell viability and complex preparation processes, which has driven the development of cell-free alternatives such as MSC-Exos. These 30–150-nm vesicles contain bioactive proteins, lipids, and RNAs that mediate the therapeutic effects of MSCs [99], while offering superior stability, standardized production, and non-immunogenicity. These advantages position MSC-Exos as a promising fertility treatment.

Overview of MSC-derived exosomes

Wolf discovered extracellular vesicles (EVs) in 1967 through the ultracentrifugation of platelets, and named them ‘platelet dust’ [100]. Ratajczak et al. in 2006 then demonstrated that EVs were mediators of intercellular communication [101].EVs are a general term for membrane vesicles secreted by cells and mainly include three categories: exosomes (Exos), microvesicles, and apoptotic bodies [102]—of which Exos are a core functional subgroup of EVs due to their ability to deliver signals precisely and also because of their biocompatibility in intercellular communication. This has received extensive attention in fields such as regenerative medicine [103], and EVs have great potential for gene delivery, disease diagnosis, intracellular communication, drug delivery, and biomarker-driven therapies. Exosomes are approximately 30–150 nm in diameter and are composed mainly of biomolecules such as proteins, lipids, mRNAs, and miRNAs, which are important carriers of intercellular communication [99]. The chief process underlying EV generation is the invagination of the plasma membrane to form endocytosed vesicles. This is followed by the inward budding of the endosomal membrane to form multivesicular bodies (MVBs) that contain multiple intraluminal vesicles (ILVs), and the eventual fusion of the MVBs with the plasma membrane releases the ILVs as Exos [104, 105]. MVBs have two fates: if they merge with lysosomes, their contents are degraded by hydrolytic enzymes; alternatively, if they fuse with the plasma membrane, they are released into the extracellular milieu as exosomes through the process of exocytosis [105, 106]. Characteristic molecules that comprise MVBs are tetraspanin proteins (e.g. CD63, CD9, and CD81), lysosome-associated membrane proteins (LAMPs-1, -2, and − 3), and late endosomal markers (e.g. MHC class II molecules, flotillin, and HSP70), which aid in exosomal sorting and recognition [107, 108].

Exosomal formation is controlled by two mechanisms, the endosomal sorting complex required for transport (ESCRT)-dependent pathway and the ESCRT-independent pathway. The ESCRT-dependent pathway is the principal way by which the ESCRT complex (which contains core proteins such as ALIX and TSG101) controls the production of MVBs by recognizing ubiquitinated cargoes and facilitating the formation of ILVs [109, 110]. Additionally, the non-ESCRT-dependent pathway is where sphingomyelinase 2 hydrolyses sphingomyelin to produce ceramides or assists in the transport of cargoes via tetraspanins (e.g. CD63) that facilitate cargo sorting and exosomal release [111].

Exosomes carry signaling molecules such as proteins, lipids, and nucleic acids and are important carriers of intercellular communication, and their composition is highly dependent upon the function of the cells from which they originate (e.g. MSCs) [112, 113]. Exosomes are also enriched in a variety of functional proteins, including four transmembrane proteins (CD9, CD63, CD81, and CD82) that are involved in cell adhesion, signaling, and membrane stability; heat shock proteins (e.g. HSP70 and HSP90) that assist in protein folding and are involved in the immune response; MVB-forming proteins (e.g. apoptosis-linked gene 2 interacting protein X [ALIX], tumor susceptibility gene 101 [TSG101], and syntenin) that regulate multivesicular body formation and exosomal release; and membrane transport proteins (e.g. membrane association proteins and Rab proteins) that assist in the fusion of the exosome with the target cell [114]. Kim et al. [115] adopted a proteomic approach to identify 730 functional proteins in human MSC-Exos, many of which promote cell division and growth factor secretion, providing a molecular basis for MSC-Exo functioning. Lipids are the principal components of exosomal membranes, primarily ceramide, cholesterol, and arachidonic acid [111, 116, 117]. Lipids not only make up the membranous structure, but they also participate in sorting contents and signaling. Exosomes ferry genetic information such as microRNA (miRNA), long non-coding RNA (lncRNA), DNA, and messenger RNA (mRNA), and they act by regulating gene expression in the recipient cells. miRNAs in the exosomes of immune cells, for example, regulate the immune responses of target cells [118], and lncRNAs are preferentially packaged into exosomes—for example, lncRNAs in HeLa cell exosomes enhance the viability of the cervical squamous carcinoma cell line C33A [119]. Exosomes contain mitochondrial DNA (mtDNA) that can act as an oncogenic signal to promote cancer stem cell activation (e.g. mtDNA in breast cancer cell exosomes leads to endocrine therapy resistance) [120, 121], and mRNAs are delivery-function genes (e.g. mRNAs in MSC-Exos induce growth factor secretion in recipient cells) [122] (Fig. 3).

Fig. 3.

Biogenesis, secretion and molecular composition of mesenchymal stem cell-derived exosomes (MSC-Exos). a MSC-Exos are formed through three stages: plasma membrane invagination, endosomal budding, and membrane fusion. b MSC-Exos carry proteins, lipids, and nucleic acids. (Created in BioRender by Wang, Y. (2025) (https://BioRender.com/mp4s7cd)

Exosomes play an important role in cellular communication and epigenetic regulation, but two factors limit both basic and applied research on exosomes: the simplification of exosomal extraction and the enhancement of their production, and the effective differentiation of exosomes from other EVs (especially functional microvesicles).Cell type is one of the factors that influences exosomal production, and immature dendritic cells produce a limited number of exosomes [123], while MSCs are the most abundant producers of exosomes [124]. Exosomes are currently isolated by sequential ultracentrifugation [125], density-gradient centrifugation [126], ultrafiltration [127], volumetric exclusion chromatography [128], sedimentation [125], immunoaffinity capture [129], and microfluidics [130]; each technique reflects its own advantages and disadvantages. Exosomes with scaffolding materials (e.g. collagen scaffolds [131], hydrogels [132], and electrostatic spinning [133]) used as delivery carriers can be optimized for maximal loading to significantly improve retention, prolong the duration of action, and maintain the biological activity of the exosomes.

While the recovery of tissue function at the time of injury was previously accomplished by applying cellular therapies, Kusuma et al. [134] proposed the use of exosomes and microvesicles secreted by MSCs as an alternative and revealed that this regimen of emulating the paracrine activity of MSCs was reproducible. Exosomes come from a wide range of sources, including bone marrow, adipose tissues, amniotic membranes, and the umbilical cord [135]. These exosomes are all similar in morphology and immunophenotype, and they all engage in proliferation, migration, and apoptosis [136]. However, exosomes from differing sources have distinct actions in disease treatment. Zou et al. [137] discovered that MSCs and their exosomes manifested similar miRNA-expression profiles and that the target genes of exosome-enriched miRNAs were associated with calcium channel regulation and cell junction activity—suggesting that exosomes and MSCs possess unused regulatory properties. MSC-Exos can regulate a variety of physiological processes and participate in intercellular communication, cell signaling, and tissue metabolism [138, 139]. This suggests that MSC-Exos might be applied to cell-free regenerative medicine under certain conditions. MSC-Exos are also critical to the treatment of cellular damage [140] by controlling inflammatory diseases, as they stimulate vascular regeneration, reduce fibrosis and remodeling, and enhance immune cell function [141]. Based on the aforementioned biological properties of MSC-Exos and their underlying mechanisms of action in fertility enhancement—particularly the specific molecular pathways they use for improving endometrial tolerance, restoring ovarian function, and regulating the reproductive immune microenvironment [9]. MSC-Exos have in recent years become a central research topic in the field of reproductive medicine. Given this, we herein systematically elucidate the mechanisms of action of MSC-Exos from different sources of MSCs in infertility treatment to provide novel theoretical support and a direction for translational exploration in cell-free treatment strategies of infertility (Fig. 4).

Fig. 4.

Seven key pathways (angiogenesis-related, NF-κB, PI3K/AKT, TGFβ/Smad, oxidative stress-related, Hippo, and MAPK) illustrating how mesenchymal stem cell-derived exosomes (MSC-Exos) integrate multi-target regulation to modulate the pathological processes of infertility. Abbreviations: VEGFR, vascular endothelial growth factor receptor; VEGF, vascular endothelial growth factor; LIF, leukemia inhibitory factor; avβ3, integrin alpha-v beta-3; IGF-1, insulin-like growth factor 1; TF, transcription factor; LPS, lipopolysaccharide; NF-κB, nuclear factorκB; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; PTEN, phosphatase and tensin homologue deleted on chromosome 10; mTOR, mechanistic target of rapamycin; Bcl-2, B-cell lymphoma/leukemia-2; TGFβ, transforming growth factor beta; Smads, Sma- and Mad- related proteins; ROS, reactive oxygen species; YAP/TAZ, Yes-associated protein/ transcriptional co-activator with PDZ-binding motif; MST1/2, mammalian sterile 20-like kinase 1/2; TEAD, TEA domain transcription factor; RTKs, receptor tyrosine kinases; RAS, rat sarcoma viral oncogene homolog; RAF, rapidly accelerated fibrosarcoma kinase; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase(Created in BioRender by Wang, Y. (2025) (https://BioRender.com/in2kdha)

Expression of exosomes from disparate sources in infertility

Exos, as significant mediators of intercellular communication, exhibit key regulatory functions in reproductive physiology and pathology by regulating gene and protein expression; mediating the proliferation and differentiation of GCs and follicles; supporting the growth of oocytes; and participating throughout the processes of fertilization, embryonic implantation, and embryonic development towards the successful gestation of pregnancy [142]. An in-depth analysis of the crosstalk by Exos in the secretome of the female reproductive system will also provide a theoretical foundation for the screening of diagnostic markers and the development of targeted therapeutic strategies for diseases related to the reproductive system.

Ye et al. [143] collected follicular fluid from five patients with PCOS and five healthy controls, and via miRNA high-throughput sequencing, they uncovered 124 differentially expressed miRNAs (78 upregulated and 46 downregulated) from their follicular fluid exosomes. Functional enrichment analysis of the target genes of the differentially expressed miRNAs revealed that they were principally involved in metabolic pathways such as the insulin signaling pathway, lipid metabolism, and glucose transport—suggesting that these miRNAs could be involved in the pathogenesis of PCOS by regulating metabolic processes. Follicular fluid exosomes from PCOS patients harbor different circRNA expression levels, of which hsa-circ-0006877 is processed from their low-density lipoprotein receptor (LDLR) gene, and its depletion may be associated with the higher expression of miR-1294 and cytochrome P450 family 19 subfamily A member 1 (CYP19A1). Figure 5 shows that the competing endogenous RNA (ceRNA) network of circLDLR-miR-1294-CYP19A1 may control some PCOS-related pathways such as ovarian steroidogenesis [144]. Hu et al. [145] found that exosomes from follicular fluids of PCOS patients contained small RNA sequences and could be important in driving the pathogenesis of PCOS; thus, they could be used as molecular biomarkers for future PCOS diagnoses.

Fig. 5.

miRNA-mediated intercellular communication via mesenchymal stem cell-derived exosome (MSC-Exo) secretion. a The circLDLR-mediated ceRNA network. (Reproduced with permission [144]. Copyright © 2020 Huang et al. Aging. Published by Rapamycin Press Ltd.) b Prediction of target genes of differentially expressed exosomal miRNA (Reproduced with permission [148]. Copyright © 2020 Zhou et al. Reproductive Healthcare Ltd. Published by Elsevier Ltd.)

Using RNA sequencing, Wu et al. [146] demonstrated by RNA sequencing that at least 1449 mRNAs, 938 lncRNAs, and 39 miRNAs exhibited differential expression patterns in exosomes derived from normally positioned endometrial cells, ovarian endometriotic tissues, and normal endometrial stromal cells (ESCs); of these, 61 competing ceRNAs were also reported. In addition, a recent study showed that serum levels of exosomal miR-22-3p and miR-320a were significantly elevated in patients with endometriosis, which could comprise suitable biomarkers for the diagnosis of endometriosis [147]. As illustrated in Fig. 5b, Zhou et al. [148] found that exosomal miRNAs—including those derived from ESCs of patients with endometriosis—were not expressed in the serum. Exosomal miRNAs (including hsa-miR-494-3p, hsa-miR-10b-3p, hsa-125b-2-3p, and hsa-miR-1343-3p) also retained higher levels of 12 miRNAs that were predicted to target homeobox protein A10 (HOXA10) and/or the leukemia inhibitory factor (LIF) 3’ untranslated region (UTR).

miR-223 is the most abundant miRNA in macrophage-derived exosomes, is dysregulated in patients with EM, and has been shown to contribute to the activation of M2 macrophages [149]. Exosomes from ESCs activate macrophages, polarizing them into an M2-like phenotype that subsequently promotes the progression of endometriotic lesions in mice [150]. In addition, the peritoneal macrophage-derived exosomal miR-22-3p was involved in the cell proliferation, migration, and invasion of ectopic ESCs by regulating the SIRT1/NF-κB signaling pathway [151].

There appears to be an association between sperm quality and abnormal miRNA levels in seminal plasma and exosomes as Wang et al. [152] ascertained that seven miRNAs (miR-34c-5p, miR-122, miR-146b-5p, miR-181a, miR-374b, miR-509-5p, and miR-513a-5p) were significantly reduced in azoospermia but elevated in spermiosis. Similarly, miR34b/c and miR-449 have been associated with male infertility due to impaired sperm motility [153]. We posit that these miRNAs may be used as diagnostic biomarkers for idiopathic male infertility and in therapeutic development.

Bone marrow-derived mesenchymal stem cells (BM-MSC-Exos)

Exosomes secreted by BM-MSCs operate as intercellular communication vectors, and the miRNAs they carry are significant in the restoration of ovarian function. Yang et al. [154] found that miR-144-5p in BM-MSC-Exos inhibited the apoptosis of CTX-injured GCs via the downstream target gene phosphatase and tensin homologue deleted on chromosome 10 (PTEN) that activated the PI3K/AKT signal transduction pathway. Sun et al. [155] ascertained that miR-644-5p borne by BM-MSC-Exos inhibited apoptosis of ovarian GCs by targeting p53 in cells. BM-MSC-Exos contain proteins intricately involved in inflammation, cell adhesion, fibrosis, and apoptosis—including IL13, IL1 receptor-like 2 (IL1RL2), IL5, IL-10, fibronectin 1 (FN1), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), and growth hormone 1 (GH1). In addition, the TGF β superfamily proteins growth differentiation factor 3 (GDF3) and GDF11 have been identified in BM-MSC-Exos and are shown to regulate cancer cell apoptosis [156].

BM-MSC-Exo-derived miR-340 translocates to endometrial stem cells (EnSCs) to ameliorate endometrial fibrosis, promote vascular endothelial cell proliferation, and repair damaged endometrium; however, the exact molecular mechanisms underlying these activities remain unclear [157]. Li et al. [158] found that BM-MSC-Exos appeared to negatively regulate HOXA10 through miR-196b in a rat model of TE, which in turn, promoted the expression of VEGF, LIF, and integrins—thereby increasing endometrial thickness and normalizing protein expression. miR-31 derived from ADSC-Exos also modulated endothelial cells through a mechanism related to VEGF overexpression that drove endothelial cell proliferation, migration, and tube formation [159]. ADSC-Exos contain elevated levels of miRNA-125a, miRNA-31, and miR-486-5p, and can be translocated to vascular endothelial cells to promote angiogenesis [160]. McBride et al. [161] discovered that BM-MSC-Exos promoted fibroblasts and endothelial cell proliferation and migration and stimulated dermal repair and regeneration. BM-MSC-Exos reversed the TGF-β1-induced epithelial-mesenchymal transition (EMT) in rabbit endometrial epithelial cells (EECs), and thus may have promoted endometrial repair through the TGF-β1/Smad signaling pathway. miR-21 in BM-MSC-Exos is also associated with multiple signaling pathways involved in tissue regeneration, quiescence, cellular senescence, and fibrosis; and miR-100-5p and miR-143-3p promote cellular proliferation. Specifically, miR-100-5p promoted the regenerative process through upregulation of TGF-β3, VEGFA, MMP7, and HGF [162] and blocked differentiation/decidualization, as evidenced by morphological changes and downregulation of decidualization markers that included HOXA10, insulin-like growth factor binding protein 1 (IGFBP1), prolactin (PRL), progesterone receptor isoform B (PR-B), and progesterone receptor (PR) [163]. These results reveal that microRNA delivery represents a novel therapeutic approach, potentially replacing BM-MSC-Exos in cell-free regenerative therapies [164].

Exosomes from BM-MSC-Exos and urine-derived stem cells restored spermatogenesis by promoting intercellular adhesion molecules, reducing apoptosis, and upregulating key genes associated with spermatogenesis [165, 166]. Alcayaga-Miranda et al. [167] demonstrated that Exos could also contain antimicrobial peptides and proteins with bactericidal properties. BM-MSC-Exos are involved in bacterial pneumonia, which is associated with enhanced phagocytosis of bacteria by human monocytes, reduced inflammation, and increased ATP levels in alveolar type 2 epithelial cells.

In conclusion, miRNA-mediated regulation of gene expression comprises an important basis for the therapeutic actions of BM-MSC-Exos.

Exosomes derived from ADSCs (ADSC-Exos)

Upregulation of miR-323-3p in ADSC-Exos for delivery to GCs revealed that elevated levels of miR-323-3p effectively inhibited apoptosis in GCs, suggesting that ADSC-Exos constituted a highly efficient modality for the delivery of functional molecules such as miRNAs in the course of PCOS interventions [168]. Cao et al. [169] showed that ADSC-EXOs activated the insulin receptor substrate 1/protein kinase B (IRS1/AKT) signaling pathway and enhanced liver glucose and lipid metabolism by delivering miR-21-5p to the rat livers of rats with experimentally induced PCOS. Furthermore, ADSC-EXOs targeted and inhibited B-cell translocation gene 2 (BTG2) expression [170], thereby modulating glucose and lipid metabolism and alleviating metabolic disorders. This miRNA transfer mechanism also improved the polycystic state of the ovary and enhanced the fertility of the rats. Huang et al. [170] discerned that ADSC-Exos could restore the ovarian function in POI by regulating the SMAD pathway and that knockdown of SMAD increased apoptotic genes (Fas, FasL, caspase-3, and caspase-8), as well as induced ovarian follicle loss. The miRNA-based engineering of MSC-Exos provides succinct direction for the treatment of PCOS in future basic and clinical studies.

ADSC-Exos stimulated ovarian proliferation, inhibited apoptosis, and attenuated the expression of SIRT4 and downstream genes in GCs. Ding et al. [171] demonstrated that ADSC-Exos enhanced POI ovarian function by targeting SIRT4 via miR-320a.

The expression of VEGF, LIF, integrin alpha-v beta-3 (avβ3), and IGF-1 was shown to be significantly elevated in a TE rat model in which ADSC-Exos were incorporated, and pregnancy and implantation rates were higher than in a blank group after treatment. This result indicates that ADSC-Exos promote reestablishment of endometrial function and improved fertility [172]. ADSC-Exos promoted endometrial growth in IUA rats, augmented the number of glands, attenuated endometrial fibrosis, and elevated the levels of related molecular markers such as integrins, LIF, and VEGF, which, in turn improved fertility [173]. ADSC-Exos contain key factors related to angiogenesis that help in the regeneration of injured tissues by inhibiting apoptosis and enhancing regenerative properties. For example, Shao et al. [174] found that lncRNA-MIAT in ADSC-Exos ameliorated endometrial fibrosis by targeting miR-150-5p and that overexpression of miR-150-5p significantly upregulated TGFβR1 and α-SMA and downregulated CK19.

ADSC-Exos inhibited lipopolysaccharide (LPS)-induced inflammation in endometrial cells and mouse models. Application of ADSC-Exos restored the expression of miR-21, and overexpression led to the downregulation of inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, and increased the proliferation of ESCs. ADSC-Exos inhibited LPS-induced inflammatory responses in endometrial cells both in vivo and ex vivo, possibly through the miR-21/TLR 4/Nf-κB signaling pathway [175]. ADSC-Exos were additionally effective in alleviating sepsis syndrome-induced systemic inflammation and attenuating organ damage and adverse outcomes in rats [176].

ADSC-Exos from dogs affected canine sperm structure and function during cryopreservation, initiating repair of damaged sperm and reducing ROS production, thereby improving the quality of thawed canine semen [177]. Exos were protective against sperm cryo-damage (e.g. cell membrane and DNA damage) and oxidative stress from cryopreservation, and they improved sperm parameters after thawing [178]. Support cell-derived Exos containing miR-30a-5p enhanced the proliferation and differentiation of spermatogonial stem cells (SSCs) by activating the mitogen-activated protein kinase (MAPK) signaling pathway. Intratesticular injection of 10 IU of mouse serum-derived exosomes alleviated acute inflammation by reducing the levels of nitric oxide (NO), malondialdehyde (MDA), and apoptotic gene expression; additionally, exosomes restored testosterone levels, resulting in the production of healthy spermatozoa [179].

Exosomes derived from menstrual blood-derived mesenchymal stem cells (MenSC-Exos)

Work by Mansoori et al. [180] revealed that the estrogen secretory function of GCs was enhanced by promoting mitochondrial biogenesis. However, the efficacy of MenSC-Exos in improving GC abnormalities (e.g. dysfunction and apoptosis) in PCOS patients was weaker than that observed with MenSCs, and because of the short half-life of exosomes and the short-term nature of the effects, multiple doses were required to achieve a sustained effect on GCs [181]. In addition, several studies have shown that exosomal therapy can adjust the ovarian stroma towards a more favorable microenvironment. Zhang et al. [182] demonstrated that MenSC-Exos enhanced the expression of middle lamellar adhesion proteins, collagen IV, and FN1 in POI ovaries, suggesting an effect on the regulation of the ovarian microenvironment through the promotion of primordial follicle activation and development, as well as through the inhibition of apoptosis during in vitro ovarian culture. In aged mouse models, MenSC-Exos enhanced embryo quality and quantity through the modulation of antioxidant enzymes and augmentation of pluripotency [183]. Due to the utility of exosomal therapies, exosomes are easier to store and produce [184], and we postulate that therapeutic efficacy can be bolstered by improving exosomal isolation in the future, enhancing their stability, or adding MenSCs to MenSC-Exos in combination therapy.

Davoodi et al. [185] obtained MenSC-Exos by density-gradient centrifugation, applied them to the treatment of endometriotic cells, and found that they significantly reduced the expression levels of markers related to inflammation, proliferation, migration, and angiogenesis. Their results have provided preliminary evidence of the potential of MenSC-Exos in improving EM and demonstrated the potential of exosomes to serve as a cell-free product in EM repair. Ababzadeh et al. [186] showed that MenSC-Exos and ginger root-derived exosomes (P-Exos) exhibited positive effects on gene expression and function in endometriotic cells. As plant exosomes are more readily available and less expensive, they can be considered for clinical use to improve the symptoms of endometriosis patients. Zhang et al. [187] depicted the efficacy of MenSC-Exos as consistent with that of MenSCs. MenSC-Exos effectively restored the morphology of the IUA uterus, promoted the regeneration of glands and angiogenesis, and reversed endometrial fibrosis by inhibiting the TGFβ1/SMAD3 pathway in the endometrium of IUA and simultaneously promoting SMAD1/5/8 and extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation and upregulation of bone morphogenetic protein 7 (BMP7) expression. In addition, MenSC-Exos manifest a superior local therapeutic effect relative to MenSCs that migrate in the bloodstream, comprising a promising non-cellular therapy for endometrial regeneration.

Exosomes from umbilical cord-derived mesenchymal stem cells (UC-MSC-Exos)

UC-MSCs inhibit chronic inflammation by reducing the production of inflammatory mediators such as TNF-α and IFN-γ, increasing IL-10 levels and anti-inflammatory cytokines, and decreasing apoptosis in ovarian GCs. Zhao et al. [188] determined that UC-MSC-Exos inhibited NF-κB signaling by lowering the levels of phosphorylated IκB and p65 in GCs, thereby enhancing the expression of the anti-inflammatory cytokine IL-10. Furthermore, UC-MSC-Exos suppressed the expression of the pro-inflammatory factors TNF-α and IFN-γ, mitigating the inflammation of GCs in PCOS patients. An investigation by Tang et al. [189] revealed that anti-apoptotic miRNAs from UC-MSC-Exos significantly attenuated cisplatin-induced cellular damage through the transfer of anti-apoptotic miRNAs. These authors’ miRNAs (miR-26a-5p, miR-222-3p, miR-143-3p, and let-7i-5p) in exosomes targeted and regulated apoptosis-related pathways in cells to protect GCs from the toxic effects of chemotherapeutic drugs. Ding et al. [190] found that microRNA-17-5P from UC-MSC-Exos was effective in reducing cisplatin-induced cell injury by inhibiting the expression of SIRT7 and its downstream target genes γH2AX, PARP1, and XRCC6 in order to promote the proliferation of chemotherapeutically injured human GCs and ovarian cells and to inhibit the accumulation of ROS, bolstering the fertility of mice with POI.

Furthermore, Li et al. [191] demonstrated that in a POI murine model treated with UC-MSC-Exos, the expression levels of key Hippo signaling pathway components—including Yes-associated protein (YAP), transcriptional co-activator with PDZ-binding motif (TAZ), and TEA domain transcription factor (TEAD)—were significantly elevated. This upregulation enhanced cellular proliferative capacity, promoted GC proliferation and functionality, and ultimately improved ovarian function and reproductive performance in the POI mice. Transplantation of UC-MSC-Exos also ameliorated ovarian function in POI rats by exerting anti-inflammatory effects through TNF- and IL-mediated signaling pathways, improving the local ovarian microenvironment (including cellular viability, inflammation, immunomodulation, fibrosis, and metabolism), upregulating gene clusters involved in ovarian gene repair, and promoting ovarian steroidogenesis to regulate folliculogenesis and maintain reproductive function [192].

Yang et al. [193] found that UC-MSC-Exos carried functional microRNAs (miR-146a-, miR-21-, miR-146a-5p, miR-146a-, miR-21-, and miR-21-) and expressed ROS in human GCs and ovarian cells and that these Exos inhibited ROS accumulation. miR-146a-5p and miR-21-5p targeted the PI3K/mTOR signaling pathway to activate primordial follicle development, which promoted follicle development and oocyte quality in aged female mice [194]. In addition, Zhu et al. [195] further extended our understanding of the role of UC-MSC-Exos in the protection of ovarian GCs by finding that exosomal circBRCA1 stabilized by the m6A demethylase fat mass and obesity-associated protein (FTO) attenuated oxidative stress-induced cellular damage and that this was achieved through the miR-642a-5p/Forkhead box O1 axis.

UC-MSC-Exos advance the proliferation of EECs in a dose-dependent manner through the transport of miR-21-5p. For example, Hua et al. [196] They then demonstrated that knockdown of ssc-miR-21-5p hindered AKT phosphorylation, upregulated Bax expression, and downregulated the levels of B-cell lymphoma 2 (Bcl2) and MMP9 by targeting programmed cell death protein 4 (PDCD4), inhibited the proliferative and migratory behaviour of EECs and induced their apoptosis by knocking down ssc-miR-21-5p. Shi et al. [197] noted that UC-MSC-Exos attenuated mifepristone-induced apoptosis of ESCs, that miR-7162-3p was transported by UC-MSC-Exos, and that it regulated APOL6 by targeting the 3’-UTR in ESCs. Overexpression of miR-7162-3p in UC-MSC-Exos was also executed in the cell-free treatment of endometrial injury. The synergistic effect of UC-MSC-Exos in conjunction with estrogens provides a potent alternative regenerative agent for the treatment of IUA, and it renders significantly reduced inflammation and fibrosis (via TNF-α, TGF-β, IL-1, IL-6, RUNX2, and collagen-I) and VEGF expression in IUA-treated rats. Furthermore, there was a significant increase in angiogenesis compared with the authors’ model group [198]. UC-MSC-Exos accelerated endometrial proliferation of ESCs, and a study by Lv et al. [199] further revealed that UC-MSC-Exos could be directly internalized by ESCs and that they stimulated the growth of ESCs in a dose-dependent manner—with effects occurring over a short duration. A study by Ebrahim et al. [198] revealed that a diminution in inflammatory cytokines (TNF-α, IL-1, and IL-6) and fibrotic markers (Runt-related transcription factor 2 (RUNX2), TGF-β, and collagen-I)—alone or in combination with estrogen—significantly reduced uterine adhesions in female rats.

Wang et al. [200] described UC-MSC-Exos as upregulating Bcl-2 levels, downregulating cleaved caspase-3 levels, and activating the PTEN/AKT signaling pathway to regulate proliferation and anti-apoptosis. This modulation of mifepristone-injured ESCs is promising in the cell-free treatment of endometrial injury. Liakath et al. [201] discerned that UC-MSC-Exos circumvented reproductive toxicity and preserved fertility by protecting SSCs and ecological niches during chemotherapy, providing a basis for applying UC-MSC-Exos prior to chemotherapy to improve fertility and thereby enhance future reproductive prospects.

Role of MSC exosomes from other sources in infertility

Xiao et al. [202] demonstrated that amniotic fluid-derived Exos (AF-Exos) targeted Bim and downregulated the caspase-9 gene to deliver miR-146a and miR-10a and reduce follicular atresia. It is worth noting that systemic administration of high doses of miRNAs may induce side effects in clinical applications, but due to the short half-life of miRNAs, local injection at the site of injury should reduce the side effects while maintaining therapeutic efficacy. AF-Exos restored sperm parameters such as viability, concentration, and the number of spermatogonia and spermatocytes in rats, ultimately restoring the fertility of the males. Rats receiving 40 µg of AF-Exos showed a significant increase in key regulators of germ cell genesis and function (i.e. deleted in azoospermia-like [DAZL] and DEAD-box helicase 4 [VASA]) [203].

Zhang et al. [204] showed that exosomes from amnion-derived mesenchymal stem cells (AD-MSC-Exos) reversed the chemotherapy-induced increase in cleaved caspase-3 protein expression in GCs by overexpressing miR-1246 and miR-21-5p. Their results suggest that AD-MSC-Exos can be transferred to GCs and thereby inhibiting apoptosis in GCs due to miRNAs.

Seok et al. [205] used exosomes from placenta-derived mesenchymal stem cells (PMSC-Exos) to improve ovarian function by upregulating the expression of antioxidant enzymes such as catalase and peroxiredoxin-1 (PRDX1) in ovariectomized rats. These enzymes promote mitochondrial function and reduce apoptosis by decreasing ROS levels in follicular mitochondria. Liu et al. [206] uncovered PMSC-Exo regulation of the TGF-β/Smad pathway via miR-125b-5p, miR-30c-5p, and miR-23a-3p—which repaired endometrial damage in animals and enhanced their fertility. This result provides a rationale for the cell-free treatment of IUA through exosome-based therapies.

Tabeeva et al. [207] showed that proliferation-associated proteins (e.g. EGF and PDGF) in exosomes from pluripotent stem cell-derived mesenchymal stem-like cells (PSC-MSC-Exos) activated the fibroblast MAPK/ERK pathway to promote cellular proliferation. This activity, then, stimulated angiogenic factors such as VEGF and Ang-1 in endothelial cells to form luminal structures and promote neoangiogenesis that led to some amelioration of IUA.

Summary and prospects

MSC-Exos are valuable as a cell-free therapeutic strategy in fertility enhancement, and their core mechanism of action lies in their delivery of functional cargos (e.g. miRNAs and lncRNAs) to mediate intercellular communication, thus providing a novel, low-immunogenic therapeutic strategy for reproduction-associated disorders by regulating the survival, proliferation, and differentiation of target cells (Table 1). Several animal models and preclinical studies have revealed that MSC-Exos improve reproductive dysfunction through pro-angiogenic, immunomodulatory, anti-fibrotic, and anti-oxidative stress pathways, as well as that MSC-Exos are biocompatible due to their low immunogenicity, thus avoiding the risk of allogeneic transplantation issues.

Table 1.

Summary of effective outcomes in reproductive disorders treated with mesenchymal stem Cell-Derived exosomes

Type	Source	Mechanism	Results	Ref
PCOS	ADSC- Exos	miR-21-5p activates the IRS1/AKT pathway.	Improves the polycystic status of the ovaries and increased fertility.	[169]
	MenSC-Exos	Mitigates mitochondrial dysfunction and oxidative stress.	Enhance estrogen production in granulosa cells.	[180]
	UCMSC-Exos	Targeting p-IκB/p65-NF-κB suppresses p65 nuclear translocation in GCs.	Alleviate inflammation in GCs.	[188]
POI	BM-MSC-Exos	miR-144-5p reduces atretic follicles and inhibits granulosa cell apoptosis.	Effective against CTX-induced POI.	[154]
	ADSC-Exos	Regulating the SMAD pathway to reduce the expression of follicular apoptosis genes (Fas/FasL/caspase-3/8); miR-320a targets SIRT4.	Stimulates ovarian proliferation and inhibits apoptosis.	[170, 171]
	UC-MSC-Exos	Hippo pathway promotes ovarian cell function; Exerting anti-inflammatory effects through TNF/IL pathways	Improve reproductive capacity.	[191, 192]
	AF-Exos	Via Bim targeting, caspase-9 downregulation, and miR-146a/10a delivery.	Reduce follicular atresia.	[202]
	AD-MSC-Exos	MiR-1246/21-5p overexpression attenuates chemo-induced cleaved caspase-3 in GCs	Effectively inhibits GCs apoptosis.	[204]
TE	BM-MSC-Exos	miR-340 inhibits HOXA10 and promotes VEGF, LIF, integrins.	Repair damaged endometrium.	[157]
	ADSC-Exos	miRNA-125a, etc., are transferred to endothelial cells.	Promotes angiogenesis.	[160]
	UC-MSC-Exos	Ssc-miR-21-5p inhibits AKT, upregulates Bax.	Inhibited EEC proliferation, migration, induced apoptosis.	[196]
IUA	BM-MSC-Exos	Inhibit EMT via TGF-β1/Smad pathway	Promotes cell proliferation and migration.	[161]
	ADSC-Exos	Enhance integrin, LIF, and VEGF levels.	Promotes endometrial growth, gland proliferation, and inhibits fibrosis.	[173]
	UC-MSC-Exos	Activate the PTEN/AKT pathway.	Inflammation, fibrosis, and VEGF reduced.	[196, 200]
	MenSC-Exos	Inhibit TGFβ1/SMAD3, promote SMAD1/5/8.	Promotes gland regeneration/angiogenesis and antifibrosis.	[187]
	PMSC-Exos	Regulates the TGF-β/Smad pathway through miR-125b-5p/30c-5p/23a-3p.	Repairs endometrial damage and enhances fertility.	[206]
	PSC-MSC-Exos	Activates the MAPK/ERK pathway via EGF/PDGF to promote VEGF/Ang-1 secretion.	Induces angiogenesis to ameliorate IUA.	[207]
EM	BM-MSC-Exos	miR-100-5p promotes regeneration by upregulating factors like TGF-β3.	Blocked differentiation or decidualization.	[162, 163]
	ADSC-Exos	Alleviates inflammation via the miR-21/TLR4/NF-κB pathway.	Promotes HESC proliferation and inhibits LPS-induced inflammation.	[175]
	UC-MSC-Exos	Enhances DNA synthesis and promotes cell cycle progression.	Promotes ECS.	[199]
	MenSC-Exos	Reduces markers of inflammation, proliferation, migration, angiogenesis.	Weakened the inflammation of endometriotic cells.	[185]
Female Genital Infecti–ons	UC-MSC-Exos	MicroRNA-17-5p alleviates ROS.	Restored ovarian phenotype and function.	[190]
Sperm-Related Disord-ers	AF-Exos	DAZL and VASA increased.	Promote sperm motility/concentration and spermatogenic cell proliferation.	[203]
Male Genital Infecti-ons	BM-MSC-Exos	Upregulates key genes associated with spermatogenesis.	Restored spermatogenesis.	[15, 32, 201]
	ADSC-Exos	Alleviated organ damage.	Alleviated systemic inflammation.	[33]

Abbreviations: PCOS, polycystic ovary syndrome; POI, premature ovarian insufficiency; CTX, cyclophosphamide; VEGF, vascular endothelial growth factor; LIF, leukemia inhibitory factor; EECs, endometrial epithelial cells; TE, thin endometrium; IUA, intrauterine adhesions; HESCs, human endometrial stromal cells; ECSs, endometrial stromal cells

MSC-Exos reflect a broad therapeutic prospect in ovarian dysfunctional diseases. They can improve follicular development and hormone secretion in PCOS by reducing apoptosis in GCs through miR-323-3p and other miRNAs [168], elevating estrogen secretion, and restoring the follicular microenvironment by downregulating fibrotic genes (e.g. CTGF) [182]. MSC-Exos are also involved in metabolic regulation; for example, adipose mesenchymal stem cell exosomes are able to improve reproductive dysfunction through miR-21-3p and other anti-fibrotic genes [184]. Exos activate the IRS1/AKT pathway via miR-21-5p to improve hepatic metabolic abnormalities [169], while participating in anti-inflammatory and immune regulation. For example, they inhibit the NF-κB signaling pathway, reduce the expression of pro-inflammatory factors (TNF-α, IL-6, and IL-1β), and improve the function of GCs [175]. In POI, MSC-Exos restore hormone levels, elevate serum AMH and estrogen, reduce circulating FSH, and improve the estrous cycle [72]. MSC-Exos protect ovarian reserve function, deliver molecules such as miR-144-5p to inhibit apoptosis in GCs (e.g. they diminish caspase-3 and upregulate Bcl-2 levels), and activate the SMAD pathway (SMAD2/3/5) to promote follicular development and luteal formation [154]. MSC-Exos also repair chemotherapeutic damage and attenuate ovarian disturbances from medically induced POI by regulating Treg cells and related cytokines (such as TGF-β and IFN-γ) [188].

MSC-Exos repair endometrial damage through multiple pathways—combining high efficiency and safety in TE and IUA treatments—and are expected to comprise a novel strategy that will replace traditional surgery and drug therapy. They can promote endothelial proliferation and tolerance in TE by increasing endothelial thickness through downregulation of HOXA10 and upregulation of VEGF, LIF, and integrins via miR-196b [158]. Furthermore, they contain pro-angiogenic factors such as miR-31 that drive endothelial cell proliferation and angiogenesis [159]; inhibit apoptosis by activating the PDCD4/AKT pathway via miR-21-5p (thereby downregulating Bax and upregulating Bcl2); and promote epithelial cell proliferation [196]. MSC-Exos improve pregnancy outcome, and preclinical studies have shown that MSC-Exo treatment increases embryonic implantation and pregnancy rates [172]. In the treatment of IUA, MSC-Exos can exert anti-fibrotic effects and promote tissue regeneration by inhibiting the EMT and reverse fibrosis through the transforming growth factor beta 1/ Sma- and Mad-related proteins(TGF-β/Smad) pathway [161], and they can also reduce collagen deposition and upregulate VEGF and LIF, thereby promoting the regeneration of glands and blood vessels [172]. MSC-Exos exert anti-apoptotic actions (in the upregulation of Bcl-2 and downregulation of caspase-3) through the PTEN/AKT pathway; combine with estrogen to synergistically reduce inflammation and fibrosis by attenuating TNF-α and TGF-β expression [200]; inhibit the TGFβ1/SMAD3 pathway by activating SMAD1/5/8 and ERK1/2; promote BMP7 expression; and repair endometrial structure [187]. Compared with traditional surgery (e.g. transcervical resection of adhesions), MSC-Exo treatment reduces the recurrence of postoperative adhesions [198].

Tubal infertility (e.g. EM and tubal occlusion) constitutes one of the major causes of female infertility, and traditional treatments (i.e. laparoscopic surgery and antibiotics) have shown a high recurrence rate and limited efficacy [81, 208]. MSC-Exos have therefore emerged as a therapeutic strategy for tubal repair due to their anti-inflammatory, anti-fibrotic, pro-reparative, and immune-modulatory effects. MSC-Exos attenuate endometriotic cell inflammation; enhance proliferation, migration, and angiogenesis [150]; regulate immune cells; promote macrophage conversion from the M1-type to the M2-type; and inhibit T-cell over-activation [81]. Furthermore, Exos promote epithelial repair by inhibiting apoptosis (by downregulating caspase-3) via miRNAs such as miR-17-5p; enhance epithelial cell proliferation in the fallopian tubes [82]; and inhibit LPS-induced inflammatory responses through attenuated miR-21/TLR 4/Nf-κB signaling [175].

Male infertility involves a variety of etiological factors, including abnormal sperm quality, reproductive tract infections, and oxidative stress injury [14]. Traditional treatments (hormone therapy, surgery, or antibiotics) harbor limitations, and MSC-Exos encompass a promising therapeutic strategy due to their reparative, antioxidant, anti-inflammatory, and antimicrobial properties. They can also improve sperm quality, protect spermatozoa after freezing, attenuate damage from ROS, and increase sperm viability after thawing (e.g. in the canine semen assay) in sperm-related disorders [177]. MSC-Exos improve spermatogenesis, restore sperm concentration and viability, and upregulate key factors in germ cells [203]. Furthermore, miR-30a-5p-containing MSC-Exos activate the MAPK pathway and promote SSC proliferation and differentiation [179]. They advance antioxidant effects, and mouse serum Exos have been shown to reduce NO and MDA (markers of oxidative stress), restore testosterone levels, and reduce apoptosis [178]. MSC-Exos also shield post-chemotherapy fertility, attenuate the toxicity of chemotherapeutic drugs (e.g. alkylating agents) on SSCs, maintain spermatogenesis, and are suitable for fertility preservation of pediatric cancer patients [201]. These vesicles show anti-inflammatory effects, and modified Exos (e.g. loaded with antimicrobial peptides) can enhance bactericidal effects [167].

Despite their promising future, the clinical translation of MSC-Exos still faces many challenges. For example, their current production is limited and relies on traditional culture methods, and a suitable method needs to be developed to achieve large-scale production and to establish a strict quality-control system. The specific targets of signaling pathways governing exosomal cargoes (e.g. miRNAs and proteins) have not been fully elucidated yet, restricting their precise therapeutic design. The in vivo retention time of exosomes is also relatively short, and this may affect their long-term efficacy. A majority of extant studies entail short-term observations without long-term data support, and most existing data are based on in vitro experiments or mouse models; only a few early clinical trials fully consider the assessment of exosomal safety. The efficacy endpoints of MSC-Exos (such as pregnancy rate and live-birth rate) in humans also still need to be verified by large-sample, multicenter, randomized controlled trials. The pathways that regulate exosomal miRNA/mRNA should be clarified in the future through multi-omics technologies combined with functional validation to clarify their core molecular mechanisms of action. Fast and efficient isolation technologies should be developed, and internationally unified standards need to be formulated to regulate the isolation, identification, and activity assessments of exosomes to ensure batch consistency. We expect to conduct early clinical trials to assess the efficacy and safety of MSC-Exos on target patients by using pregnancy rate and live-birth rate as the principal endpoints, explore the synergistic effects of combined hormonal therapy, and improve therapeutic efficiency.

With continuous mechanistic explorations and technological advancements regarding exosomes, these vesicles are expected to develop into a revolutionary therapy in the field of reproductive medicine, providing new hope for millions of infertile couples. The application of exosomes will not only assist patients in achieving their fertility needs, but they will also enhance reproductive health by delaying ovarian aging and improve endometrial quality. The development of this modality will bolster patients’ overall quality of life and allow the realization of individualized treatments using MSC-Exos as molecular carriers.

Abbreviations

MSC-Exos

Mesenchymal stem cell exosomes

ARTs

Traditional assisted reproductive technologies

IUI

Intrauterine insemination

IVF-ET

In vitro fertilisation-embryo transfer

ICSI

Intracytoplasmic sperm injection

OHSS

Ovarian hyperstimulation syndrome

MSCs

Mesenchymal stem cells

PCOS

Polycystic ovary syndrome

POI

Premature ovarian insufficiency

POF

Premature ovarian failure

TE

Thin endometrium

IUA

Intrauterine adhesions

CS

Chronic salpingitis

PID

Pelvic inflammatory disease

EM

Endometriosis

BM-MSCs

Bone marrow-derived mesenchymal stem cells

GCs

Granulosa cells

ROS

Reactive oxygen species

FSH

Follicle-stimulating hormone

IGF-1

Insulin-like growth factor-1

ADSCs

Adipose-derived mesenchymal stem cells

VEGF

Vascular endothelial growth factor

LDLR

Low-density lipoprotein receptor

MenSCs

Menstrual blood-derived mesenchymal stem cells

T1D

Type 1 diabetic

UC-MSCs

Umbilical cord-derived mesenchymal stem cells

HGF

Hepatocyte growth factor

TGF-β1

Transforming growth factor-beta 1

IL-1β

Interleukin-1 beta

TNF-α

Tumour necrosis factor-alpha

AFSCs

Amniotic fluid-derived mesenchymal stem cells

AD-MSCs

Amnion-derived mesenchymal stem cells

PMSCs

Placenta-derived mesenchymal stem cells

PSC-MSCs

Pluripotent stem cell-derived mesenchymal stem-like cells

EVs

Extracellular vesicles

Exos

Exosomes

MVBs

Multivesicular bodies

ILVs

Intraluminal vesicles

ESCRT

Endosomal sorting complex required for transport

miRNA

MicroRNA

LncRNA

Long non-coding RNA

mRNA

Messenger RNA

ceRNA

Competing endogenous RNA

ESCs

Endometrial stromal cells

EECs

Endometrial epithelial cells

ADSC-Exos

Exos derived from ADSCs

MAPK

Mitogen-activated protein kinase

MenSC-Exos

Exosomes derived from menstrual blood-derived mesenchymal stem cells

ERK

Extracellular signal-regulated kinase

UC-MSC-Exos

Exosomes from umbilical cord-derived mesenchymal stem cells

AF-Exos

Amniotic fluid-derived Exos

Author contributions

Conceptualization; data curation; formal analysis; writing—original draft: Yuhan Wang. Data curation; formal analysis: Ruiling Wang, Yunxia Zhao, Nannan Han. Formal analysis: Xiaojing Han. Investigation: Tong Shi, Yamei Liu, Nian Zhao, Sufen Li. Conceptualization; writing—review and editing: Shaoqiang Wang. Conceptualization: Niannian Li. Conceptualization; funding acquisition; writing—review and editing: Yuchun Zhu. All authors have read and approved the final version of the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (No.32261160571), the National Natural Science Foundation of China (No.82230052),and the Weifang Municipal Health Commission (No.WFWSJK-2024-143).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.